Contents

Goal

Design and fabricate a single copy vector in which BioBricks devices can be characterized. To date most characterization work has been done in low or high copy vectors which have several issues including

Copy number is uncertain or variable making it difficult to infer PoPS per DNA copy.

At high copy, devices place a high metabolic load on the cell thereby altering host physiology and observed device behavior.

The proposed solution to these two problems is to characterize devices at single copy in the cell. Obviously, such a vector will vary between 1 and 2 copies per cell over the cell cycle but nevertheless will hopefully present an improvement over the current situation. The advantage of using a single copy plasmid rather than simply integrating the device into the genome is that a separate plasmid offers some isolation from the host and makes moving the device between different host strains slightly easier.

a high copy origin in the multiple cloning site to enable easy purification of the vector

strong terminators flanking the BioBricks insertion site

no loxP or cos insertion sites or Tn7 attachment sites?

I can't think of an obvious use of these sites unless we want to build in the capability for integrating onto the genome. However, wouldn't it make more sense to build in recombination capabilities onto a higher copy number vector than this?

no blue-white screening?

inclusion of a lacZα fragment would restrict its use as a part

multiple versions with different antibiotic resistance markers

no selection system for mammalian cells

VF2 and VR sites

unique tag near but outside the cloning sites for identification during sequencing. (from Randy)

resistance markers

orient the ampicillin antibiotic resistance cassette on the reverse strand from the BioBricks insertion site

every plasmid should have AmpR plus another resistance marker.

need to include a terminator downstream of the antibiotic resistance cassette (use a terminator from the original BioBricks plasmids)

include a sequence with translational stops in all frames flanking each side of the MCS

apparently when you sequence, the first 30 bp or so are really bad (see also the talk page) but there can also be a bad spot at around base pair 80. So the verification primers should be about 100bp away from the multiple cloning site.

the plasmid origin transcripts should be oriented in the forward direction such that readthrough from the origin can't affect the insert.